Tag Archives: calcium

Signal mediators at induction of heat resistance of wheat plantlets by short-term heating

Yu. V. Karpets, Yu. E. Kolupaev, T. O. Yastreb

V. V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

The effects of functional interplay of calcium ions, reactive oxygen species (ROS) and nitric oxide (NO) in the cells of wheat plantlets roots (Triticum aestivum L.) at the induction of their heat resistance by a short-term influence of hyperthermia (heating at the temperature of 42 °С during 1 minute) have been investigated. The transitional increase of NO and H2O2 content, invoked by heating, was suppressed by the treatment of plantlets with the antagonists of calcium EGTA (chelator of exocellular calcium), lanthanum chloride (blocker of calcium channels of various types) and neomycin (inhibitor of phosphatidylinositol-dependent phospholipase C). The rise of hydrogen peroxide content, caused by hardening, was partially suppressed by the action of inhibitors of nitrate reductase (sodium wolframate) and NO-synthase (NG-nitro-L-arginine methyl ester – L-NAME), and the increasing of nitric oxide content was suppressed by the treatment of plants with the antioxidant ionol and with the scavenger of hydrogen peroxide (dimethylthiourea). These compounds and antagonists of calcium also partially removed the effect of the rise of plantlets’ heat resistance, invoked by hardening heating. The conclusion on calcium’s role in the activation of enzymatic systems, generating reactive oxygen species and nitric oxide, and on the functional interplay of these signal mediators at the induction of heat resistance of plantlets by hardening heating is made.

Ca(2+) accumulation study in isolated smooth muscle mitochondria using Fluo-4 AM

O. V. Kolomiets, Yu. V. Danylovych, G. V. Danylovych, S. O. Kosterin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua

The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presen­ce of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating­. The apparent activation constant of Ca2+ accumulation is 53.9 ± 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation medium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.

Reactive oxygen forms and Ca ions as possible intermediaries under the induction of heat resistance of plant cells by jasmonic acid

Yu. V. Karpets, Yu. E. Kolupaev, T. O. Yastreb, O. I. Oboznyi,
M. V. Shvydenko, G. A. Lugova, A. O. Vayner

Dokuchayev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

The participation of reactive oxygen species (ROS) and calcium ions in realization of influen­ce of exogenous jasmonic acid (JA) on the heat resistance of wheat coleoptiles has been investigated­. Influence of 1 µM JA caused the transitional intensifying of generation of superoxide anion-radi­cal (O2•–) and hydrogen peroxide in coleoptiles with the maximum within 15-30 minutes after the treatment beginning. Within the first hour after the beginning of coleoptiles treatment with JA the increase of superoxide dismutase (SOD) activi­ty was noted. Later on (within 5-24 hours after the treatment beginning) there was the lowering of ROS generation by coleoptiles of experimental variant, and the SOD activity approached the control value. Intensifying of generation of superoxi­de radical induced by JA was suppressed by the antioxidant ionol and was partially levelled by imidazole (inhibitor of NADPH-oxidase), EGTA (chelator of extracellular calcium) and lanthanum chloride (calcium channels blocker). Pretreatment of coleoptiles with the ionol, imidazole, EGTA and LaCl3 also partially removed the effect of increase of their resistance to the damaging heating caused by exogenous JA. It is supposed that the ROS gene­rated with participation NADPH-oxidase, which activity depends on the receipt of calcium ions from extracellular space in the cytosol, are involved in realization of physio­logical effects of JA.

Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells

Yu. V. Danylovych, S. A. Karakhim, H. V. Danylovych, O. V. Kolomiets, S. O. Kosterin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua

We demonstrated using Ca2+-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes’ inner mitochondrial membrane by a protonophore carbonyl cyanide m-chlorophenyl hуdrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3′-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane’s electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hуdrazone causes substantial increase in fluorescence of Ca2+-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca2+-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.

NAADP-sensitive Сa(2+) stores in permeabilized rat hepatocytes

S. V. Bychkova1, T. I. Chorna2

1Ivan Franko National University of Lviv, Ukraine;
2National Centre for Biological Sciences, Tata Institute
of Fundamental Research, Bangalore, India;
e-mail: s.bychkova@gmail.com

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a nucleotide that is potent to release calcium from intracellular stores in different cell types. NAADP was shown to target specific type of intracellular store namely endolysosomal system or acidic store. Despite intense studies, its effect on endoplasmatic reticulum (ER) still remains to be elucidated. The main aim of our work was to investigate NAADP-sensitive store in permeabilized rat hepatocytes monitoring the level of Ca2+ inside intracellular organelles using chlorotetracycline (CTC). We have shown that NAADP triggered changes of stored Ca2+ in rat hepatocytes are dependent on concentration of EGTA-Ca2+-buffer in cell incubation medium, i.e. the higher is the EGTA concentration in incubation medium the smaller or absent is the effect of NAADP. Besides, the effect of NAADP was more pronounced upon cells pretreatment with the inhibitory concentration of ryanodine (100 µM). This might suggest that the effect of NAADP is dependent on ER luminal calcium. We have also found that NAADP-evoked Ca2+ release in permeabilized hepatocytes is sensitive to nigericin, bafilomycin A and thapsigargin. Additionally, NAADP triggered changes in stored Ca2+ were completely abolished by NED-19 as antagonist of NAADP.

Reactive oxygen species and stress signaling in plants

Yu. E. Kolupaev, Yu. V. Karpets

V. V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

Data on the basic processes and the compartments, involved in formation of reactive oxygen species (ROS) in plant cells, are generalised. The features of structure and regulation of NADPH-oxidase as the one of main enzymatic producers of ROS are characterized. The two-component histidine kinases, ROS-sensitive transcript-factors, ROS-sensitive protein kinase and redox-regulated ionic channels are discussed as the possible sensors of redox-signals in plant cells. The interaction between ROS and other signal mediators, in particular nitric oxide and calcium ions, is discussed. The ROS role as the signal mediators in the development of plant resistance to hyperthermia, osmotic shock and other abiotic stressors is analyzed.

Role of Ca ions in the induction of heat-resistance of wheat coleoptiles by brassinosteroids

Yu. E. Kolupaev1, A. A. Vayner1, T. O. Yastreb1, A. I. Oboznyi1, V. A. Khripach2

1V. V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru;
2Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus;
e-mail: khripach@iboch.bas-net.by

The involvement of Ca2+ into the signal transduction of exogenous brassinosteroids (BS) (24-epibrassinolide – 24-EBL and 24-epicastasterone – 24 ECS) causing the increase of heat resistance of the cells of wheat (Triticum aestivum L.) coleoptiles was investigated using calcium chelator EGTA and inhibitor of phosphatidylinositol-specific phospholipase C – neomycin. Twenty-four-hour treatment of coleoptile segments with 10 nM solutions of 24-EBL and 24-ECS led to a transient increase in the generation of superoxide anion radical by cell surface and the subsequent activation of superoxide dismutase and catalase. Pretreatment of coleoptiles with EGTA and neomycin depressed to a considerable extent these effects and leveled the increase in heat resistance of wheat coleoptiles that were caused by BS. Possible mechanisms of involvement of calcium signaling into the formation of reactive oxygen species in plant cells and induction of heat resistance of plant cells by the action of exogenous BS have been discussed.